1. Field of the invention
This invention relates to a novel enzyme which catalyzes a dephosphorylation and protein. The protein having an enzyme activity of the present invention supplies a phosphoric acid which is particularly necessary for of an oosperm at a growing period thereof and is a substance capable of exterminating injurious insects by inhibiting a growth effect of the oosperm or an activity thereof.
2. Prior art
Biology has made remarkable progress of late. As a result, various molecules explaining morphogenesis have been identified as of today. There have been known growth factors which drastically induce morphogenesis, adhesion factors which mediate between cells, and the like.
However, there are many morphologies of differentiation which cannot be explained by the known growth factors or adhesion factors. Among these, researches on protease which take notice of development have scarcely been carried out so that the present inventor aimed the function of protease at the development process. Protease can make reaction irreversible by decomposing a protein so that it can be considered that it would be inevitable for development of an organism having a property of channeling or direction. Thus, it can be considered that if the function of the protease at the development stage can be clarified, a novel viewpoint can be obtained for understanding the development.
Up to this day, as a protease which participates in the development, Sarcophaga peregrina cathepsin L has been found. The sarcophaga peregrina cathepsin L is shown to be essential for morphogenesis at metamorphosis stage of complete metamorphosis insects. In the complete metamorphosis insects, there exists. The imaginal disk is existing in a larva body and differentiated to an imago tissue by stimulation of ecdysone which is a metamorphosis hormone at the period of chrysalis.
It has been known that an imaginal disk was extracted from a larva body and morphogenesis to an imago tissue can be reproduced in an in vitro culture system in which the imaginal disk cultured in the presence of ecdysone. There has been also reported by using the above culture system that it is essential for differentiate the imaginal disk to an imago leg to secrete cathepsin L out of cells by the induction of ecdysone and to decompose a protein on a basement membrane of the imaginal disk. This demonstrates that intracellular lysosome enzyme cathepsin L has an important function in morphogenesis in addition to the function of decomposing an unnecessary protein in a cell.
The action of cathepsin L at metamorphosis stage has now been elucidated with a certain extent but the action of cathepsin at a development stage other than the metamorphosis stage has not yet been known. It is important to elucidate the function of cathepsin L at the development stage since it makes possible to control development by promoting of inhibiting et al. Thus, the present inventor has analyzed gene expression and protein expression of cathepsin L.
As a result, I have obtained an interesting knowledge particularly at the initial stage of embryogenesis. It has been clarified that cathepsin L exists as maternal mRNA and a precursor protein in a non-fertilized egg and exists as a matured protein from the initial stage of embryogenesis. That is, with regard to fertilized eggs and non-fertilized eggs of sarcophaga peregrina (Non-fertilized eggs are collected by separating imagoes immediately after eclosion to male and female, grown in separate cages and collected from abdomen of female after 5 days from eclosion using a pincette in an insect saline. Fertilized eggs are collected from female which is bred in a cage simultaneously with male. After eclosion, embryo at the fifth day is called as "early", at the 7th days as "middle" and at the 9th days as "late".), presence of cathepsin L is tested. The results are shown in FIG. 1. In FIG. 1, 50 .mu.g of homogenate protein is used in respective lanes, and Lane 1 shows a non-fertilized egg, Lane 2 early egg embryo, Lane 3 middle egg embryo and Lane 4 late egg embryo. An arrow shows the position of a matured type cathepsin L (35 kDa). As can be clearly seen from Lanes 1 and 2 in FIG. 1, the presence of a matured type cathepsin L is confirmed from early embryo.
Early embryogenesis of complete metamorphosis insects may look like different from the other organisms. At the early stage of embryogenesis, however, embryo has not yet differentiated to the respective tissues and many reserved life phenomena which are similar to the other species can be found. For example, determination of dorso-ventical axis is made by a concentration gradient of a growth factor along with the body axis, and it has been reported that it is completely the same mechanism between drosophila (fruit fly) and African clawed toad.
Accordingly, if the function of cathepsin L can be clarified at the early embryogenesis, there is a great possibility of showing the function of protease common in embryogenesis not only in insects, but also in a common organisms.
From the above fact, the present inventor has conducted identification of an intravital substrate at the early embryogenesis was subjected to cloning to clarify an important function of cathepsin L at the early embryogenesis which can be considered to be common to a living body. It has also been desired to prepare an antibody to the protein.
The present inventor tried to identify an intravital substrate at the early embryogenesis to clarify the function of cathepsin L in the early embryogenesis. And analysis of localization of a substrate protein and cloning of cDNA are carried out as mentioned below.
(1) A protein with a molecular weight of 100 kDa on SDS-PAGE which can be considered to be an intravital substrate of cathepsin L at the early embryogenesis in a non-fertilized egg homogenated protein is determined.
(2) From the analysis of localization in the early embryo, it is found that the 100 kDa protein exists at the center portion of embryo in which cells have not yet been formed and cathepsin L is similarly localized.
(3) By cloning of cDNA which encodes the 100 kDa protein, the 100 kDa protein is found to be a novel protein.
(4) An antibody to the 100 kDa protein is prepared.